CN101636175A - Biomarkers for Neurological Conditions - Google Patents

Abstract

Found low-molecular-weight (LMW) peptide, its be such as the A Zihai Mo's disease (Alzheimer ' s Disease, AD), the indication of the little neurological conditions such as hemorrhage of cognitive disorder and brain.The existence of the peptide of LMW described in the evaluating patient samples is the effective means that detects neurological conditions and the described disease process of monitoring.Described LMW peptide is particularly useful for detecting neurological conditions during in early days under the situation of not taking the invasive program.

Description

Biomarkers for Neurological Conditions

Cross References to Related Applications

This application claims priority to US Provisional Application Serial No. 60/855,749, filed November 1, 2006, which is incorporated herein by reference.

technical field

The present invention provides a method for diagnosing a neurological condition in a patient, comprising obtaining a biological sample from said patient, and evaluating said sample at least one selected from the group consisting of peptides having an amino acid sequence of SEQ ID NO: 1-440 The abundance of the biomarkers of the panel, wherein the abundance of the at least one biomarker is indicative of a neurological condition.

Background technique

Alzheimer's disease (AD) is a progressive brain degenerative disease mainly associated with aging. AD is one of several conditions that cause a gradual loss of brain cells and is one of the likely leading causes of dementia. The clinical manifestations of AD are characterized by loss of memory, cognition, reasoning, judgment and orientation. Mild cognitive impairment (MCI) is usually the first identified stage of AD. As the disease progresses, motor, sensory, and language abilities are also affected, until total impairment of multiple cognitive functions occurs. These cognitive losses occur gradually but usually lead to severe impairment and eventually death over a period of 3 to 20 years.

Early diagnosis of AD has many benefits, including extra time to make choices that optimize quality of life, less anxiety about the unknown, a good time to benefit from treatment, and more time to plan for the future. However, reasonable non-invasive methods of diagnosing AD are not available.

Alzheimer's disease is characterized by two main pathological observations in the brain: neurofibrillary tangles (NFTs) and beta-amyloid plaques composed mainly of aggregates of fragments called Aβ peptides. Individuals with AD exhibit characteristic β-amyloid deposits as well as neurofibrillary tangles in the brain (β-amyloid plaques) and in cerebral blood vessels (β-amyloid angiopathy). Neurofibrillary tangles occur not only in Alzheimer's disease, but also in other dementia-induced conditions. Autopsies (currently the only authoritative method for diagnosing AD) have found that a large number of these lesions are found in areas of the human brain that are critical to memory and cognition.

There is an urgent clinical need to develop diagnostic markers that can detect early AD, especially MCI stage. Despite progress in imaging β-amyloid (Lopresti et al., J. Nucl. Med. (2005) 46:1959-1972), there are no clinically available Serum biomarkers for AD. To date, there are no effective biomarkers for diagnosing severe neurodegenerative disorders or monitoring progress (Castano et al., Neurol. Res. (2006) 28: 1155-163).

Despite decades of effort to use proteomic techniques to discover blood markers for AD, it is possible that putative highly specific AD markers are obscured by small amounts of diseased tissue and expected to be rapidly cleared and metabolized. Representing very low abundance, the process for identifying useful markers is extremely slow. In addition, researchers have avoided the presence of resident proteins such as albumin in the blood proteome, which can be present in concentrations millions of times higher than low-abundance target biomarkers. Start a blood study. To this end, researchers have focused on studying cerebrospinal fluid (CSF) as a target fluid for AD biomarkers (see Zhang et al., J. Alzheimer's Disease (2005) 8 : 377-3386). However, the CSF approach limits clinical application to routine screening. Furthermore, cerebrovascular blood circulation perfuses AD lesions with high efficiency, especially in the case of amyloid angiopathy.

Contents of the invention

In one aspect, a method for diagnosing a neurological condition in a patient is provided, comprising obtaining a biological sample from the patient, and assessing at least one of the samples selected from the group consisting of peptides having the amino acid sequence of SEQ ID NO: 1-440. Abundance of the biomarkers of the population, wherein the abundance of the at least one biomarker is indicative of a neurological condition. In one embodiment, the abundance of the biomarker is higher than the abundance of the biomarker in the control sample. In another embodiment, the abundance of the biomarker is lower than the abundance of the biomarker in the control sample.

The method may further comprise, prior to the assessing step, collecting low molecular weight peptides from said sample to generate at least one fraction comprising said peptides. A biomarker can be a low molecular weight protein complexed with a carrier protein. In another embodiment, low molecular weight proteins are additionally purified from said carrier protein. In another embodiment, the low molecular weight protein is digested and optionally sequenced. In one embodiment, the biological sample is blood, serum or plasma. In another embodiment, the evaluating step comprises an analysis selected from the group consisting of: mass spectrometry, such as tandem mass spectrometry (MS MS); immunoassay, such as enzyme-linked immunosorbent assay (ELISA); immuno-mass spectrometry ; and a suspension bead array. The method may also comprise obtaining neuroimages of cerebral microvascular lesions, which may optionally be obtained using susceptibility weighted imaging, perfusion weighted imaging, and magnetic resonance spectroscopy.

The neurological condition can be Alzheimer's disease (AD), mild cognitive impairment (MCI), stable mild cognitive impairment (stable MCI), progressive mild cognitive impairment (PMCI), vascular dementia (VD ), angiopathy black hole, cerebroamyloid angiopathy (CAA) and cerebral microbleeds. In one embodiment, there is provided a method for diagnosing Alzheimer's disease in a patient, comprising obtaining a biological sample from said patient, and evaluating said sample at least one selected from the group consisting of peptides having the amino acid sequence Abundance of biomarkers of the cohort: SEQ ID NO: 1, 3-13, 15, 16, 21, 22, 24-28, 31-33, 37-44, 56-59, 66-68, 93-101 , 111-128, 143-153, 156-1170, 172-183, 263-279, 310-335, 348, 355-359, 362, 363, 365, 372, 373, 376-402, 406-426, and 436 - 44, wherein the abundance of the at least one biomarker is indicative of Alzheimer's disease. Biomarkers, on the other hand, are peptides related to metabolic pathways or cellular processes. In other aspects, biomarkers are related to inflammation, estrogen activity, pigment epithelium-derived factor (PEDF), vitamin D metabolism and bone mineralization, coagulation and platelet activity, complement cascade, acyl peptide hydrolysis Enzyme (APH) activity, vitamin A and thyroxine, phospholipase activity, globulin activity, glycosylated or glycosylated, protease inhibition, keratin and related proteins, heme degradation, pyruvate metabolism, calcium-related proteins, Defensin, gelsolin, vitronectin, profibrin, thromboxane, peroxiredoxin, alcohol dehydrogenase, apolipoprotein, iron and copper metabolism or NMDA receptor-related proteins of peptides.

In another aspect, there is provided a method for diagnosing mild cognitive impairment in a patient, comprising obtaining a biological sample from the patient, and evaluating at least one of the samples selected from the group consisting of peptides having the amino acid sequence Abundance of biomarkers: SEQ ID NO: 2, 4, 14, 17, 23, 29, 34, 45-55, 60-65, 69-92, 102-110, 129-142, 154, 155, 171, 184-191, 193-226, 248-279, 281-320, 333, 336-347, 349-354, 360, 361, 364, 366-371, 374, 375, 403-405 and 427-435, of which The abundance of the at least one biomarker is indicative of mild cognitive impairment.

In another aspect, a method for diagnosing cerebral microbleeds in a patient is provided, comprising obtaining a biological sample from the patient, and evaluating at least one peptide selected from the amino acid sequence having SEQ ID NO: 441-452 in the sample The abundance of the biomarkers of the group consisting, wherein the abundance of the at least one biomarker is indicative of cerebral microbleeds.

In some embodiments, the methods of the invention comprise, prior to the evaluating step, collecting low molecular weight peptides from said sample to generate at least one fraction comprising said peptides. The size of a low molecular weight peptide may eg be less than 50 KDa, less than 25 KDa or less than 15 KDa. The method may also comprise diagnosing low molecular weight peptides. The digestion can be achieved using enzymatic or chemical means. In one example, the peptides can be digested using trypsin.

In other aspects, antibodies specific to biomarkers of neurological conditions are provided, as well as kits comprising at least one such antibody for use in detecting a neurological condition in a patient. Antibodies can be, for example, monoclonal or polyclonal antibodies, and can also be chimeric, humanized or human antibodies.

Other objects, features and advantages will be apparent from the following embodiments. The detailed description and specific examples are provided for illustrative purposes only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Furthermore, the examples will describe the principles of the invention and it is not contemplated that in all examples the applications of the invention will be specified where it would be obvious to those skilled in the art.

Description of drawings

none

Detailed ways

Low molecular weight (LMW) peptides indicative of neurological conditions have been discovered from the lineage of proteins bound to carrier proteins such as albumin. Assessing the presence of said LMW peptides in patient samples is an efficient way to detect neurological conditions and monitor the progression of said diseases, eg during treatment. LMW peptides are especially useful for detecting neurological conditions during their early stages. LMW peptides are especially useful for detecting AD, MCI and cerebral microbleeds.

LMW peptides as biomarkers can be detected using a variety of methods known in the art. For example, antibodies can be used in immunoassays to detect the presence of biomarkers. Exemplary immunoassays include, for example, ELISA, radioimmunoassay, immunofluorescence assay, "sandwich" immunoassay, western blot, immunoprecipitation assay, and immunoelectrophoretic assay. In other aspects, bead, array, microarray, etc. assays can be used in the detection of LMW peptides. Exemplary assays include, but are not limited to, suspension bead assays (Schwenk et al., "Determination of binding specificities in highly multiplexed microbead assays for antibody proteomics") bead-based assays for antibody proteomics), "Molecular and Cellular Proteomics (Mol. Cell Proteomics), 6(1): 125-132 (2007)), antibody microarrays (Borrebaeck et al., " High-throughput proteomics using antibody microarrays: an update," Expert Rev. Mol. Diagn. 7(5): 673-686( 2007)), aptamer arrays (Walter et al., "High-throughput protein arrays: prospects for molecular diagnostics," Trends Mol Med. 8 (6): 250-253 (2002)), affybody arrays (Renberg et al., "Affybody molecules in protein capture microarrays: multidomain ligands and different Evaluation of detection formats (Affibody molecules in proteincapture microarrays: evaluation of multidomain ligands and different detection formats), "Journal of Proteome Research (J.Proteome Res.) 6(1): 171-179(2007)) and reversed-phase arrays (VanMeter et al., "Reverse-phase protein microarrays: application to biomarker discovery and translational medicine," Expert Rev. Mol. Diagn.) 7(5):625-633 (2007)). All of these publications are incorporated herein by reference.

In another example, the biomarkers of the invention can be detected using mass spectrometry (MS). An example of such a method is tandem mass spectrometry (MS/MS), which involves multiple mass selection or analysis steps, usually separated by some form of fragmentation. Most of these analyzes use electrospray ionization followed by two-stage mass selection: the first stage (MS1) selects the mass of the intact analyte (precursor ion); and the second stage, after fragmenting the precursor ion by collision with gas atoms, (MS2) Specific precursor ion fragments are selected to collectively generate a selected reaction monitoring array. In one embodiment, collision-induced dissociation is used to generate a set of fragments from specific peptide ions. Fragmentation methods primarily generate cleavage products that break along peptide bonds. Due to the ease of fragmentation, the observed fragment masses can be compared to a database of predicted masses for known peptide sequences. A number of different algorithms have been described to identify peptides and proteins from tandem mass spectrometry (MS/MS) data, including peptide fragment fingerprinting (SEQUEST, MASCOT, OMSSA, and X!Tandem), peptide resequencing ( PEAKS, LuteFisk, and Sherenga) and sequence tag based searching (SPIDER, GutenTAG).

Likewise, multiple reaction monitoring (MRM) can be used to identify biomarkers of the invention in patient samples. This technique applies MS/MS methods such as trypsinization of an input sample, followed by MS to assign and sample selected ions, enabling better analyte selection by tracking the exact m/z ions representing trypsin fragments of the analyte. objective and decentralized. The method can be performed multiple times so that multiple ions can be measured at one time, thereby providing an antibody-free method for analyte measurement. See, e.g., Andersen et al., Molecular & Cellular Proteomics, 5.4:573-588 (2006); Whiteaker et al., J. Proteome Res. ) 6(10): 3962-75 (2007). Both publications are incorporated herein by reference.

In another example, nanoflow reverse-phase liquid chromatography-tandem mass spectrometry can be used to detect the biomarkers of the invention. See, eg, Domon B, Aebersold R. Science, 312(5771):212-7 (2006), which is incorporated herein by reference. Using this method, one obtains peptide fragments, typically by trypsinization, and generates mass spectra of the fragments, which are then compared to databases such as SEQUEST for protein identification.

In another aspect, the biomarkers of the invention can be detected using immunomass spectrometry. See for example Liotta L et al., J Clin Invest., 116(1):26-30 (2006); Nedelkov, Expert Review of Proteomics Rev. Proteomics), 3(6):631-640 (2006), which is incorporated herein by reference. Immuno-mass spectrometry provides a means to rapidly determine the exact size and identity of peptide biomarker isoforms present in patient samples. When developing a high-throughput diagnostic assay, a single drop of patient blood, serum, or plasma can be used in a high-density matrix of microcolumns or wells filled with immobilized polyclonal antibodies against peptide tags composite substrates. All isoforms of the peptide containing the epitope are captured. The captured analyte population, including analyte fragments, is directly eluted and analyzed by a mass spectrometer, such as MALDI-TOF MS. The presence of specific peptide biomarkers at accurate mass/charge (m/z) positions can be used as a diagnostic test result. Analysis can be performed rapidly by simple software that determines the presence or absence of a series of ion peaks at specified m/z positions.

In another example, the biomarkers of the invention can be detected using standard immunoassay-based methods in which fragment-specific antibodies are used to measure and document the presence of diagnostic fragments. See, for example, Naya et al. "Evaluation of precursor prostate-specific antigen isoform ratios in the detection of prostate cancer." Endocrine Oncology ( Urol Oncol.) 23(1): 16-21 (2005). In addition, other immunoassays, such as ELISA, are well known to those skilled in the art (Maeda et al., "Blood tests for asbestos-related mesothelioma," Oncology 71:26 -31(2006)), Microfluidic ELISA (Lee et al., "Microfluidic enzyme-linked immunosorbent assay technology," Adv.Clin.Chem. ) 42:255-259 (2006)), nanocantilever immunoassay (nanocantilever immunoassay) (Kurosawa et al., "Quartz crystal microbalance immunosensors forenvironmental monitoring , "Biosensors and Bioelectronics (Biosens Bioelectron), 22(4): 473-481 (2006)) and plasmon resonance immunoassay (Nedelkov), "Surface plasmon resonance Development of surface Plasmonresonance mass spectrometry array platform, "Analytical Chemistry (Anal.Chem.) 79(15): 5987-5990(2007)). All publications are incorporated herein by reference.

In another example, the biomarkers of the invention can be detected using electrochemical methods. See, eg, Lin et al., Anal. Sci. 23(9): 1059-1063 (2007).

In one embodiment, the LMW peptides are collected from the biological sample prior to the evaluating step. For example, 100 μl of serum can be mixed with 2×SDS-PAGE Laemmli Buffer (containing 200 mM DTT), boiled for 10 minutes and loaded on a preparative pool containing a 5 cm long 10% acrylamide gel ( Prep Cell) (type 491 prep cell, Bio-Rad Laboratories, CA)). Electrophoresis was performed at a constant voltage of 250V. After the bromophenol blue indicator dye was eluted from the system, the LMW peptides and proteins migrated from the gel and were trapped in the dialysis membrane in the elution chamber. These molecules can be eluted with the same composition buffer with Tris-glycine running buffer at a flow rate of 400ml/min and collected in aliquots over 10 minutes.

Alternatively, LMW peptides can be collected from a sample using a capture particle comprising a molecular sieve moiety and an analyte binding moiety, as described in U.S. Patent Application No. 11/2006, filed September 27, 2006, incorporated herein by reference. as described in No. 527,727. Briefly, either the molecular sieve portion or the analyte binding portion, or both, comprise cross-linked regions of altered porosity, or pore size sufficient to exclude high molecular weight molecules.

In another embodiment, LMW peptides are digested prior to detection, thereby reducing peptide size. The digestion can be performed using standard methods well known in the art. Exemplary treatments include, but are not limited to, enzymatic and chemical treatments. The treatment can result in partial as well as complete digestion. An example of enzymatic treatment is trypsinization.

The biomarkers of the invention are particularly useful for detecting neurological conditions during their early stages, such as when the conditions are still associated with MCI or PMCI, or for detecting cerebrovascular lesions, such as cerebral microbleeds. For illustration, mild cognitive impairment (MCI) cases are classified under the Mayo Clinic criteria for classification as MCI-Multiregional Impairment (MCI-MCDI) with the following characteristics: i) Corrected Logical Memory testing or history presenter's report and memory impairment as determined by CDR = 0.5. ii) normal activities of daily living; iii) normal general cognitive function; iv) abnormal memory at a certain age as measured by standard scores and education; v) total CDR of 0.5 and no dementia; vi) no obvious vascularity past history of insulin-requiring diabetes or uncontrolled high blood pressure. Meanwhile, stable mild cognitive impairment (stable MCI) is based on a sum of several assessments (Sum of boxes) = 0.5-3.5, CDR logical memory impairment (where at least one assessment indicates logical memory impairment), not always in MCI A range of neuropsychological testing and clinical judgment. Progressive Mild Cognitive Impairment (PMCI) indicates a patient's sum score ≥ 3.5 on two occasions, neuropsychological testing in full agreement with the CDR, raw scores of logical memory down to zero, and clinical judgment.

The abundance of a biomarker can be measured by detecting the biomarker as described above and comparing the amount of the biomarker to a control. The abundance of biomarkers is an indicator of a neurological condition. If a biomarker is "less abundant than a control," then the biomarker is present in the test sample in a significantly lower amount than in the control sample. If a biomarker is "more abundant" than a control, then the biomarker is present in a significantly higher amount in the test sample than in the control sample. For example, the difference can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% %, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000% or higher. A control can be a sample from a normal patient or a patient in a known disease state, or an equivalent thereof. For example, controls can be from patients suffering from AD, MCI or cerebral microbleeds. A control can also be a standard amount or a known amount of a reference peptide.

The detected neurological condition can be, for example, Alzheimer's disease (AD), mild cognitive impairment (MCI), stable mild cognitive impairment (stable MCI), progressive mild cognitive impairment (PMCI), vascular dementia (VD), vascular black hole, cerebroamyloid angiopathy (CAA) and cerebral microbleeds. Conditions and activities described herein refer to their commonly accepted definitions unless otherwise indicated. For example, as described in more detail in the Examples, cognitive impairment was defined according to the Mayo clinical criteria.

In another embodiment, the biomarkers are peptides associated with metabolic pathways or cellular processes. In other embodiments, the biomarkers are related to inflammation, estrogen activity, pigment epithelium-derived factor (PEDF) vitamin D metabolism and bone mineralization, coagulation and platelet activity, complement cascade, acyl peptide hydrolase (APH) activity, Vitamin A and thyroxine, phospholipase activity, globulin activity, glycosylated or glycosylated, protease inhibition, keratin and related proteins, heme degradation, pyruvate metabolism, calcium-associated proteins, defensins, gelsolin , vitronectin, profibrin, thromboxane, peroxidoreductase, alcohol dehydrogenase, apolipoprotein, iron and copper metabolism or peptides related to NMDA receptor-related proteins.

In one aspect, more than one biomarker can be assessed simultaneously. For example, at least 2, at least 5, at least 10, at least 20, at least 30, at least 50, at least 75, at least 100 biomarkers are assessed in the method. Analysis of more than one biomarker can increase diagnostic accuracy.

The methods of the invention can be used in combination with neuroimaging techniques to detect neuropathy and cerebral microvasculopathy associated with neurological conditions. For example, neuroimaging can be used to detect cerebral microbleeds associated with cognitive impairment. Focal decreased signal intensity secondary to hemosiderin remnants can be detected using magnetic resonance imaging. These spots on MR images are called "signal void", "susceptibility artifact", "black hole", "dot", "microbleed" , "old microbleed (OMB)", "multifocal signal loss lesion" or "microhemorrhage (MH)". Collectively, these spots are called small hypointensities (SH) and are associated with AD and MCI (Cordonnier et al. Neurology (2006) 66: 1356-1360; Werring et al. Brain (2004) 127:2265-2275). Appropriate MR imaging techniques include gradient refocused echo T ₂ * (GRE-T ₂ ) and susceptibility-weighted imaging ( _SWI )

Neuroimaging methods that detect metabolic changes in the brain can also be used in conjunction with the biomarkers of the invention. Alterations in these systems associated with neurological conditions can be analyzed using, for example, MR spectroscopy, which detects differences in neurotransmitters such as glutamate, glutamine, and gamma-aminobutyric acid (GABA). These metabolic changes can be correlated with cognitive decline and biomarker abundance.

Antibodies specific for the biomarkers of the invention can be readily prepared using methods well known in the art (see, J. Sambrook, EF Fritsch and T. Manitsch .Maniatis), Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, pp. 18.7-18.18, 1989). For example, the biomarkers of the invention can be readily prepared using an automated peptide synthesizer. Immunogens such as (peptide) _n -KLH (n=1-30) are then injected into immunocompetent animals in complete Freund's adjuvant, followed by suspension in incomplete Freund's adjuvant The same immunogen was injected into the immunocompetent animals twice successively, and splenocytes were collected 3 days after the intravenous boost of the antigen. Harvested splenocytes were then fused with Sp2/0-Agl4 myeloma cells and culture supernatants of the resulting clones were analyzed for anti-peptide reactivity using direct binding ELISA. The superior specificity of the antibodies produced can be tested by using peptide fragments of the original immunogen.

In certain embodiments, one or more antibodies directed against a biomarker of the invention are provided in a kit for use in a diagnostic method. The kits may also include reagents, instructions, and other products for performing the diagnostic methods.

In other aspects, the biomarkers and antibodies of the invention are useful for discovering novel aspects of neurological conditions, such as those described herein.

The following examples are for illustrative purposes only and should not be construed as limiting. Additionally, each reference disclosed herein and throughout this application is hereby incorporated by reference in its entirety.

example

Example 1. Background and Patient Overview

The study recruited 103 participants from the community (75 MCI individuals and 28 cognitively normal individuals). Among the initial 75 MCI individuals, 20 were excluded due to various reasons not related to dementia, and currently 55 remain for further research. According to the clinical dementia grading scale (Clinical Dementia Rating, CDR) total score ≥ 3.5 provided by the NINCDS-ADRDA standard, 17 of them became demented in the observation period of 0.5 to 4.1 years (15% annual conversion rate) (Sch Schafer et al. Alzheimer Dis Assoc Disord. (2004) 18:219-222; McKhann et al. Neurology. (1984) 34:939- 944). Among the 28 cognitively normal individuals, 4 had progressed to MCI type, and 2 of them had obvious SH detected by SWI. Two MCI cases are currently on the verge of dementia, one of which has overt SH. SWI brain imaging has shown that 7 of 17 individuals with dementia and progressive cognitive impairment had increased and "large" amounts (n > 5) of SH. The progressive increase in SH in the lobes followed by localization in cortical-subcortical patterns makes the diagnostic pattern consistent with "probable CAA" (Knudsen et al. Neurology. (2001 ) 56:537-539). This observation is the first prospective evidence of a subgroup of sporadic late-onset dementias temporally associated with increased SH in the typical pattern of CAA.

Individual choice:

After screening 1348 community-based individuals at a published memory clinic, 28 elderly "controls" and 75 individuals with MCI were eligible for the study using inclusion and exclusion criteria defined by the Mayo Clinic (Petersen RC) et al., Arch Neurol. Mar (1999) 56:303-308). Individuals were continuously assessed using sequential cognitive (twice per year) and radiological (once per year) procedures over a period of 4.1 years (range 0.5 to 4.10 years, with a mean total follow-up of 2.3 ± 1.2 years and a total of person-years of follow-up of 241.7). All subjects gave informed consent and all studies were approved by the Loma Linda University Institutional Review Board. The complete drug treatment, medical and smoking history of all individuals were obtained and the thyroid function, serum B12 content and ApoE genotype of all individuals were clarified.

Normal individuals: (n=28)

All "control subjects" had no objective or subjective memory deficits and were within normal limits on neuropsychological testing (total CDR of 0, CDR memory component of 0 and CDR sum score of 1 or below baseline). The CDR sum score was used as a measure of cognitive performance. (107)

MCI Individuals: (n=75)

All MCI cases were classified as MCI-Multiregional Impairment (MCI-MCDI) in the Mayo Clinical Classification Criteria, which had the following characteristics: i) as determined by the corrected logical memory test or the presenter's report of the history and CDR=0.5 Memory impairment; ii) normal activities of daily living; iii) normal general cognitive function; iv) abnormal memory at a certain age as measured by standard scores and education; v) total CDR of 0.5 and no dementia; and vi ) with no past history of significant vascular problems, insulin-requiring diabetes, or uncontrolled hypertension. According to examination, 20 MCI individuals were excluded for various reasons: 2 cancers, 1 co-morbidity, 2 claustrophobia, 9 lack of nursing support/relocation, 5 loss of interest and 1 bit for pacemaker.

cognitive test:

All cognitive assessments were performed by the same team of neuropsychologists during the 4-week MR assessment period and reassessed at approximately 6-month intervals. A total of 476 cognitive tests have been administered, with some individuals undergoing as many as 9 assessments. Cognitive test package includes videotaped CDR plus the following: Logical Memory I, II; North American Adult Reading Test; Verbal Fluency: Phonology and Semantics; Wisconsin Card Sorting Test ); Trail Making Test A&B; Boston Naming Test; Draw-A-Clock; Depression Features Battery Version II; Geriatric Depression Scale.

Radiological and cognitive assessment results were reviewed twice a month. In rare cases, if cognitive testing and neurologic examination indicate conditions other than AD such as frontotemporal dementia, progressive supranuclear palsy, primary progressive aphasia development, the individual was removed from the study. Neuropsychological test results were flagged as abnormal if they appeared below >1.5 standard deviations (SD) on the normative data according to age and education. The diagnosis of dementia was based on clinical judgment (consensus meeting), NINCDS-ADRDA criteria, and CDR sum score (SOB) ≥ 3.5. (107)

Cognitive processes and current neuropsychological (NP) classification for each group:

Over the past 4.1 years, the cognitive processes of the groups have been carefully monitored and a 5-grade classification has been developed (Table 5). This category serves as a matrix for the co-analysis of MR and proteomic study results. Particular attention will be paid to MCI and control cases that have progressed to cognitive loss (MCI), "dementia" or "progressive MCI" under observation.

Table 5.5 Level Cognitive NP Classification

Normal: Sum score = 0 to 0.5. CDR memory = 0, subject to clinical judgment. There were some occasional abnormalities during neuropsychological testing. Logical memory is always normal. Unstable normal (U-normal): sum score ≤ 1, but variable. An indication of some CDR memory impairment. Tendency to up- or down-regulate based on clinical judgment. Neuropsychological testing revealed moderate abnormalities with improvement or regression. clinical judgment. MCI: Sum score = 0.5-3.5 across several assessments. CDR Logical memory impairment, with impairment of logical memory in at least one of the assessments. Neuropsychological testing is not always covered by MCI. clinical judgment. Unstable MCI (U-MCI): Sum score changed from 0.5 to 3.5. Neuropsychological testing is consistent with the sum score. Considerable impairment of logical memory. Shows downward tendency. clinical judgment. Progressive MCI (PMCI) or mild AD [to be confirmed]: Sum score ≥ 3.5 on two occasions. Neuropsychological testing was consistent with CDR. Logical memory raw scores as low as zero. clinical judgment.

The above scores were derived after examining the results of multiple NP assessments. Individuals with only one assessment at baseline were classified as normal or MCI.

Table 6. 476 times NP evaluation form

Significant fluctuations in cognitive operations were found in the unstable normal and unstable MCI groups. The unstable MCI group had a cognitive status (CDR=3.5) of the onset of dementia but could be improved to 3.0 with drug treatment. Complete drug treatment histories have been obtained for all groups.

Table 7. Baseline NP (initial two categories) and current NP status according to Table 5

Table 7 provides the current NP status for each group using a 5-level classification as derived from the entrance classification (normal or MCI). Progressive normal to MCI was noted and 10 cases of MCI had moved to U-Normal and Normal, and 25 cases of MCI had moved to U-MCI (8) and PMCI (17). Human experiments were designed to measure changes in MR and proteome during dementia development.

Materials and methods

Collection of low molecular weight proteins through preparative pools

100 μl of serum was mixed with SDS-PAGE loading buffer, boiled for 10 minutes and loaded onto the prep pool (Bio-Rad, CA). After 2 hours of electrophoresis, low molecular weight proteins migrated out of the gel and eluted into collection tubes.

Nano flow reversed-phase liquid chromatography-tandem MS (nanoRPLC-MS/MS)

The protein eluted from the preparation pool was further passed through the detergent clean-upmicro kit ProteoSpin (Norgen, Canada) to remove interferences in the elution buffer. SDS for mass spectrometric analysis. Cleaned proteins were reduced with 10 mM DTT, alkylated with 50 mM iodoacetamide and digested with trypsin (from Promega) overnight at 37°C. The tryptic-digested peptides were further purified by solid-phase separation cartridge (Sep-Pak cartridge) (Waters, MA) and analyzed by using a linear ion trap mass spectrometer (LTQ, ThermoElectron). , San Jose, California (San Jose, CA)) reversed-phase liquid chromatography nanospray tandem mass spectrometry for analysis. A 100 μm internal diameter (i.d.) x 10 cm long fused silica capillary (Polymicro Technologies, Phoenix, AZ) with a laser-drawn tip in the separation column was internally filled with a 5 μm, 200 angstrom pore size C18 resin (Michrom BioResources, CA) slurry. After sample injection, the column was washed with mobile phase A (0.4% acetic acid) for 5 min, and the column was washed at 250 nL/min using a linear gradient from 0% mobile phase B (0.4% acetic acid, 80% acetonitrile) to 50% mobile phase B. Peptides were eluted for 30 minutes followed by a linear gradient of 50% mobile phase B to 100% B for an additional 5 minutes. The LTQ mass spectrometer was operated in data-dependent mode, where each full MS scan was followed by 5 MS/MS scans, where the 5 most abundant molecular ions were dynamically selected for collision-induced solution using a normalized collision energy of 35%. From (CID).

An ETD method using a thermoelectric LTQ instrument (Thermo LTQ instrument) can also be used. In contrast to traditional collision-induced dissociation (CID), the ETD method (Syka et al. Proc. Natl. Acad. Sci. U.S.A. (2004) 101: 9528-9533) in MS - Fragmentation of peptides by electron transfer in MS analysis. ETD has been shown to be more effective than CID in providing easier-to-interpret MS-MS sequence data from larger, higher charge state peptides, including intact small proteins, as well as peptides with post-translational modifications (PTMs). (Coon et al. Proc. Natl. Acad. Sci. U.S.A. (2005) 102:9463-9468). The novel combination of CID and ETD analysis can enhance the productivity of peptide identification.

Example 2: Serum proteome analysis

Separation of LMW proteins

In the first serum proteome study A, 100 μL whole serum sample aliquots were prepared for high-performance liquid chromatography/mass spectrometry (LC- MS) analysis. For subsequent studies B and C, a proteome subset consisting of low molecular weight (LMW) proteins was prepared from each serum sample to reduce the complexity of the protein mixture. The resulting LMW proteins were separated by SDS-PAGE and the proteins were visualized by Coomassie staining.

For Study B, samples consisted of serum samples pooled from 14-15 individuals (Control, MCI and PMCI). Using a modified LMW separation, serum proteins with a molecular weight of 25 kDa were collected and separated by SDS-PAGE.

For Study C, serum samples from 5 individuals who had progressed from control to MCI (1 sample) and from MCI to PMCI were prepared for LMW proteins and LC-MS was performed using a thermoelectric hybridization LTQ-Orbitrap mass spectrometer analyze. This represents the state of the art in the field of MS technology and offers several advantages over LTQ, such as excellent high mass resolution and mass accuracy of the acquired precursor peptide molecular ion spectra.

Data analysis and results.

MS-MS spectra were searched for matches against the public human protein database (NCBI) using the SEQUEST search algorithm. The results of Study A identified only abundant serum proteins. The results led to a focus on low molecular weight (LMW) serum proteins (Study B). A cutoff of 50 kDa was insufficient to reduce protein complexity, and TCA protein precipitation caused unacceptable protein loss. Therefore, a pooled sample of a relatively large number (14) of individual individual serum samples per group was used for the high quality analysis of Study B. This study compared controls to LMW proteins identified in MCI and PMCI samples/individual groups. This qualitative analysis identifies candidate biomarkers (proteins of varying abundance). The aim of Study C was to identify LMW serum proteins with different abundances that were associated with the diagnosis of progression from MCI to PMCI (4 individuals; 4 pairs of samples) and control progression to MCI (1 individual; 1 pair of samples). Analysis of these 10 samples yielded the identification of over 500 proteins. Within individual groups, there were no significant differences in apoE genotype among individuals.

Candidate biomarker proteins were identified by comparing the number of tandem mass spectra (MS2 scans) that matched the peptide sequence corresponding to the source protein in a database against which the data was searchable. Enzymatic digestion of a higher abundance protein will yield a greater number and more abundant peptides relative to a lower abundance protein, and these peptides will generally result in more matching MS2 spectra. In this way, the number of MS2 spectra (termed "spectrum count") is an approximate measure of the relative abundance of proteins in a mixture (Analytical Chemistry, 76(14), 4193-4201 (2004)). The evaluation of candidate proteins that differ in abundance focuses on proteins that produce a 50% or greater difference in spectral counts in one set of samples relative to another set of samples.

The results of the study are presented in Tables 8-10.

Example 3: Detection of cerebral microbleeds

At two locations (Detroit MRI Institute for Biomedical Research (DMRI) and Loma Linda University (LLU)) but currently primarily at LLU by raters who are members of programs using the same protocol and Clinical status unknown) SH counts were performed independently. Review the presence of SH in SWI filtered phase images of one 2mm slice at a time. During data review, images of all magnitudes, ie high-pass (HP) filtered phase images and contrast-enhanced SWI magnitude images, were used. Images were placed side-by-side to identify SH, and HP filtered phase images were used to label them with the review above and below to examine vascular connections. As shown in Figure 2, a slice may contain more than one SH, each SH is then highlighted with a different color border. Any slices exhibiting SH appearance in previous slices are no longer recounted. SHs were assigned section and sequence numbers, size (1-3, 3-5, >5 mm O.D.), and anatomical location. Microaneurysms cannot be identified using blood in and/or in the periphery of the vessel wall because blood collection in microaneurysms produces an apparently null signal. Subarachnoid and sulcal vascular spaces, symmetrical focal basal ganglia signal loss were not counted.

Biomarkers identified as associated with cerebral microbleeds are presented in Table 11.

Example 4: Further evaluation of biomarkers

The biomarkers of the invention can be further assessed using a variety of methods. In addition to traditional biovalidation analysis, mass spectrometry methods can also be used. One method of validation is Western assay of serum samples using commercially available antibodies specific for the candidate protein. If the antibody is not commercially available, it can be readily prepared using methods well known in the art and disclosed herein.

In addition, biomarkers can be further assessed using triple quadruple mass spectrometry (TQMS) technology. The technique uses multiple reaction monitoring (MRM), which consists of (1) detecting and selecting molecular ions using a first quadrupole; (2) fragmenting these ions in a second quadrupole; and (3) The ion composition of a small number of known fragments is detected in the third quadrupole. The molecular weight of the analyte and the relative abundance of fragment ions are obtained by analysis, which can characterize the analyte structure and chromatographic elution time (LC/MS). Modern TQMS instruments offer advanced MRM capabilities with higher resolution and accurate mass measurements, fast electronics for switching between large numbers of selected analytes and monitored fragment masses, and ease of use. Inherent advantages of LC/TQMS include high detection sensitivity, large dynamic detection response range, and the ability to incorporate stable isotope-labeled target analyte synthetic analogs (which allows good quantitative performance) (Anderson, Molecular and Cellular Proteomics (Mol. Cell. Proteomics) (2006) 5: 573-588; Frewen et al. Analytical Chemistry (Anal. Chem.) (2006) 78: 5678-5684).

Such studies can be enhanced with the use of spiked internal standards as well as synthetic analogs of isotopically labeled biomarkers, as in the discovery phase. In addition, autosamplers and other methods can be used to increase throughput (eg, plate-based sample peptide enrichment and removal prior to LC/MS).

Claims (58)

1. method that is used to diagnose patient's neurological conditions, it comprises:

A) obtain biological specimen from described patient; With

B) at least a abundance that is selected from the biomarker of the group that forms by the peptide of aminoacid sequence in the described sample of assessment with SEQ ID NO:1-440,

The abundance of wherein said at least a biomarker is the indication of neurological conditions.

2. method according to claim 1, the abundance of wherein said biomarker is greater than the abundance of biomarker in the check sample.

3. method according to claim 1, the described abundance of wherein said biomarker is less than the abundance of biomarker in the check sample.

4. method according to claim 1, it is additionally contained in before the described appraisal procedure, gathers low molecular weight peptide to produce the part that at least one comprises described peptide from described sample.

5. method according to claim 1, wherein said biomarker are and the compound low molecular weight protein of carrier protein.

6. method according to claim 5, wherein said low molecular weight protein are further from described carrier protein purification.

7. method according to claim 6, wherein said low molecular weight protein is through digestion and optional through order-checking.

8. method according to claim 1, wherein said biological specimen are blood, serum or blood plasma.

9. method according to claim 1, wherein said appraisal procedure comprise the analysis that is selected from the group that is become by mass spectrography, immunoassay, immunity-mass spectrography and suspension beadlet array group.

10. method according to claim 9, wherein said immunoassay are enzyme-linked immunosorbent analysis (ELISA).

11. method according to claim 9, wherein said mass spectrography comprise tandem mass spectrometry (MSMS).

12. method according to claim 1, wherein said neurodegenerative disease is selected from the group that is made up of following:

A) A Zihai Mo's disease (Alzheimer ' s disease);

B) mild cognitive impairment;

C) stablize mild cognitive impairment;

D) carrying out property mild cognitive impairment;

E) vascular dementia;

F) angiopathy black hole;

G) cerebral amyloid angiopathy; With

H) little hemorrhage.

13. method according to claim 1, it further comprises the neuroimaging that obtains the cerebral microvascular pathological changes.

14. method according to claim 13, wherein said neuroimaging is obtained by the method that is selected from by the following group that forms:

A) magnetosensitive sense weighted imaging; With

B) Magnetic Resonance Spectrum.

15. a method that is used to diagnose patient's A Zihai Mo's disease, it comprises:

A) obtain biological specimen from described patient; With

B) at least a abundance that is selected from the biomarker of the group that forms by peptide in the described sample of assessment: SEQ ID NO:1,3-13,15,16,21,22,24-28,31-33,37-44,56-59,66-68,93-101,111-128,143-153,156-1170,172-183,263-279,310-335,348,355-359,362,363,365,372,373,376-402,406-426 and 436-44 with following aminoacid sequence;

The abundance of wherein said at least a biomarker is the indication of A Zihai Mo's disease.

16. method according to claim 15 wherein saidly is labeled as following peptide:

(i) with inflammation-related and have the aminoacid sequence of SEQ ID NO:28,368-373 and 390-401;

(ii) relevant with the bone mineralising and have the aminoacid sequence of SEQ ID NO:331 and 427-435 with vitamin D metabolism;

(iii) relevant and have the aminoacid sequence of SEQ ID NO:24,102-110,157-165,350-354 and 376-389 with blood coagulation and biologically active pdgf;

(iv) relevant and have an aminoacid sequence of SEQ ID NO:117-120,129-138,139-142 and 330 with complement cascade;

(v) relevant and have the aminoacid sequence of SEQ ID NO:76-92,144-153 with globulin activity;

(vi) through glycosylation/relevant and have the aminoacid sequence of SEQ ID NO:23,50-55,46-49,174-183,193-201 and 376-389 with glycosylation;

(vii) suppress relevant and have the aminoacid sequence of SEQ ID NO:50-55,193-201 and 376-389 with protease;

(viii) relevant and have the aminoacid sequence of SEQ ID NO:154-155,202-319 and 336-338 with keratin and associated protein;

(ix) relevant with hemachrome degradation and have the aminoacid sequence of SEQ ID NO:93-98;

(x) relevant with the acetone acid metabolism and have the aminoacid sequence of SEQ ID NO:363;

(xi) relevant with the calcium associated protein and have an aminoacid sequence of SEQ ID NO:14,372-373 and 402;

(xiii) relevant with alexin and have the aminoacid sequence of SEQ ID NO:355-358;

(xii) relevant with gelsolin and have the aminoacid sequence of SEQ ID NO:166-170;

(xiv) relevant with vitronectin and have the aminoacid sequence of SEQ ID NO:436-440;

(xv) relevant with factor and have the aminoacid sequence of SEQ ID NO:360;

(xvi) relevant with thrombostondin and have the aminoacid sequence of SEQ ID NO:405;

(xvii)) relevant with peroxidating reductase or alcoholdehydrogenase and have the aminoacid sequence of SEQ ID NO:340-347;

(xviii) relevant with apolipoprotein and have the aminoacid sequence of SEQ ID NO:66-75;

(xix) relevant with ferrum and have the aminoacid sequence of SEQ ID NO:406-425 with the copper metabolism; Or

(xx) relevant with the nmda receptor associated protein and have the aminoacid sequence of SEQ ID NO:35-36.

17. method according to claim 15, wherein said biomarker are to have to be selected from by SEQ ID NO:93-98,139-142,166-170,348,355-358,402 and the peptide of the aminoacid sequence of the group that forms of 406-425.

18. method according to claim 15, the described abundance of wherein said biomarker is less than the abundance of biomarker in the check sample.

19. method according to claim 15, the abundance of wherein said biomarker is greater than the abundance of biomarker in the check sample.

20. method according to claim 15, it further comprises the neuroimaging that obtains the cerebral microvascular pathological changes relevant with the A Zihai Mo's disease.

21. method according to claim 20, wherein said neuroimaging is obtained by the method that is selected from by the following group that forms:

A) magnetosensitive sense weighted imaging; With

B) Magnetic Resonance Spectrum.

22. method according to claim 15, it is additionally contained in before the described appraisal procedure, gathers low molecular weight peptide to produce the part that at least one comprises described peptide from described sample.

23. method according to claim 15, wherein said biomarker are and the compound low molecular weight protein of carrier protein.

24. method according to claim 23, wherein said low molecular weight protein are further from described carrier protein purification.

25. method according to claim 24, wherein said low molecular weight protein is through digestion and optional through order-checking.

26. method according to claim 15, wherein said biological specimen are blood, serum or blood plasma.

27. method according to claim 15, wherein said appraisal procedure comprise the analysis that is selected from the group that is become by mass spectrography, immunoassay, immunity-mass spectrography and suspension beadlet array group.

28. method according to claim 27, wherein said immunoassay are enzyme-linked immunosorbent analysis (ELISA).

29. method according to claim 27, wherein said mass spectrography comprise tandem mass spectrometry (MSMS).

30. a method that is used to diagnose patient's mild cognitive impairment, it comprises:

A) obtain biological specimen from described patient; With

B) at least a abundance that is selected from the biomarker of the group that forms by peptide in the described sample of assessment: SEQ ID NO:2,4,14,17,23,29,34,45-55,60-65,69-92,102-110,129-142,154,155,171,184-191,193-226,248-279,281-320,333,336-347,349-354,360,361,364,366-371,374,375,403-405 and 427-435 with following aminoacid sequence;

The abundance of wherein said at least a biomarker is the indication of mild cognitive impairment.

31. method according to claim 30, the described abundance of wherein said biomarker is less than the abundance of biomarker in the check sample.

32. method according to claim 30, the abundance of wherein said biomarker is greater than the abundance of biomarker in the check sample.

33. method according to claim 30, it further comprises the neuroimaging that obtains the cerebral microvascular pathological changes relevant with the A Zihai Mo's disease.

34. method according to claim 33, wherein said neuroimaging is obtained by the method that is selected from by the following group that forms:

A) magnetosensitive sense weighted imaging; With

B) Magnetic Resonance Spectrum.

35. method according to claim 30, it is additionally contained in before the described appraisal procedure, gathers low molecular weight peptide to produce the part that at least one comprises described peptide from described sample.

36. method according to claim 30, wherein said biomarker are and the compound low molecular weight protein of carrier protein.

37. method according to claim 36, wherein said low molecular weight protein are further from described carrier protein purification.

38. according to the described method of claim 37, wherein said low molecular weight protein is through digestion and optional through order-checking.

39. method according to claim 30, wherein said biological specimen are blood, serum or blood plasma.

40. method according to claim 30, wherein said appraisal procedure comprise the analysis that is selected from the group that is become by mass spectrography, immunoassay, immunity-mass spectrography and suspension beadlet array group.

41. according to the described method of claim 40, wherein said immunoassay is enzyme-linked immunosorbent analysis (ELISA).

42. according to the described method of claim 40, wherein said mass spectrography comprises tandem mass spectrometry (MSMS).

43. the little hemorrhage method of brain that is used to diagnose the patient, it comprises:

A) obtain biological specimen from described patient; With

B) at least a abundance that is selected from the biomarker of the group that forms by the peptide of aminoacid sequence in the described sample of assessment with SEQ ID NO:441-452,

The abundance of wherein said at least a biomarker is little hemorrhage indication.

44. according to the described method of claim 43, the described abundance of wherein said biomarker is less than the abundance of biomarker in the check sample.

45. according to the described method of claim 43, the abundance of wherein said biomarker is greater than the abundance of biomarker in the check sample.

46. according to the described method of claim 43, it further comprises the neuroimaging that obtains the cerebral microvascular pathological changes relevant with the A Zihai Mo's disease.

47. according to the described method of claim 46, wherein said neuroimaging is obtained by the method that is selected from by the following group that forms:

A) magnetosensitive sense weighted imaging; With

B) Magnetic Resonance Spectrum.

48. according to the described method of claim 43, it is additionally contained in before the described appraisal procedure, gathers low molecular weight peptide to produce the part that at least one comprises described peptide from described sample.

49. according to the described method of claim 43, wherein said biomarker is and the compound low molecular weight protein of carrier protein.

50. according to the described method of claim 49, wherein said low molecular weight protein is further from described carrier protein purification.

51. according to the described method of claim 50, wherein said low molecular weight protein is through digestion and optional through order-checking.

52. according to the described method of claim 43, wherein said biological specimen is blood, serum or blood plasma.

53. according to the described method of claim 43, wherein said appraisal procedure comprises the analysis that is selected from the group that is become by mass spectrography, immunoassay, immunity-mass spectrography and suspension beadlet array group.

54. according to the described method of claim 53, wherein said immunoassay is enzyme-linked immunosorbent analysis (ELISA).

55. according to the described method of claim 53, wherein said mass spectrography comprises tandem mass spectrometry (MSMS).

56. an antibody, it is to being selected from the peptide tool specificity of the group that is made up of the peptide of the aminoacid sequence with SEQ ID NO:1-440.

57. according to the described antibody of claim 56, wherein said antibody is monoclonal antibody, polyclonal antibody or chimeric antibody.

58. a test kit that is used to detect patient's neurological conditions, it comprises at least a according to the described antibody of claim 56 with about the description of its storage or use.